Field of the Invention
The present invention relates generally to embryonic stems cells, and more specifically to a process for obtaining HLA homozygous parthenogenetic human stem cell lines for cell-based therapy.
Background Information
The first human embryonic stem cells (ESC) were derived from a blastocyst inner cell mass (ICM) obtained from a fertilized oocyte, capable of infinite division and differentiation into cells of all tissues types. The embryonic stem cell is a potentially limitless source of pluripotent cells for transplant-based cell therapies.
Human embryonic stem cells (ES) cells are pluripotent cells that can differentiate into a large array of cell types. When injected into immune-deficient mice, embryonic stem cells form differentiated tumors (teratomas). However, embryonic stem cells that are induced in vitro to form embryoid bodies (EBs) provide a source of embryonic stem cell lines that are amenable to differentiation into multiple cell types characteristic of several tissues under certain growth conditions. For example, ES cells become differentiated into neurons in the presence of nerve growth factor and retinoic acid.
Human embryonic stem cells have the potential to give significant therapeutic benefit to patients, provided that the problem of immune rejection can be solved. Embryonic stem cells that are genetically related to the recipient may overcome such rejection problems. Currently, human embryonic stem cells (hES) are derived from three sources: blastocysts remaining after infertility treatments and donated for research, blastocysts generated from donated gametes (oocytes and sperm), and the products of nuclear transfer (NT). Cadaveric fetal tissue is the only source of human embryonic germ cells (hEG). hES and hEG cells offer remarkable scientific and therapeutic possibilities, involving potential for generating more specialized cells or tissues. Ethical concerns about the sources of hES and hEG cells, however, and fears that use of NT for research could lead to use of NT to produce a human being, have fostered a great deal of public discussion and debate.
Parthenogeneic activation of mammalian oocytes may be used as an alternative to fertilization by sperm/NT to prepare oocytes for embryonic stem cell generation. Parthenogeneic activation is the production of embryonic cells, with or without eventual development into an adult, from a female gamete in the absence of any contribution from a male gamete.
Parthenogenetic activation of oocytes is a relatively simple method to create histocompatible stem cells in comparison to SCNT, because it does not require the complex equipment necessary to micromanipulate an oocyte. Parthenogenetic stem cells are produced from unfertilized oocytes and contain genetic material exclusively from the oocyte donor (the potential patient). Further, following directed cell differentiation, autologous cells may be transplanted without the threat of immune rejection. Parthenogenetic mouse MHC-homozygous stem cell lines and one parthenogenetic primate heterozygous embryonic stem cell line (Cyno-1) have already been derived and cell pluripotency has been demonstrated in these lines.
As stated above, the greatest risk posed with allogeneic tissue and organ transplantation is that of immune rejection. The degree of risk is proportional to the degree of disparity between donor and recipient cell-surface antigen-presenting proteins. In the ideal transplant, donor tissue is histocompatible with the recipient at the major histocompatibility complex (MHC). The human leukocyte antigen (HLA) system is the nomenclature designating the human MHC, and represents antigens important for transplantation. Matching donor and recipient tissue for HLA antigens reduces the chance of a cytotoxic T-cell response in the recipient, and thus greatly increases the likelihood of transplant survival.
MHC class I and II HLA haplotypes are specific sets of HLA-A, -B, -DR locus alleles inherited together from a parent. Despite a high degree of HLA polymorphism, there are only 200 common HLA haplotypes in existence within the U.S. Caucasian population. This HLA diversity, in combination with a heterozygous selection coefficient, means that the chance of finding a donor-recipient match ranges from one in 1000 to one in several million due to the unique tissue type provided by the combination of these allelic variants in the heterozygous individual.
Transplant-based stem cell therapies face the same HLA matching issues that limit solid organ allogeneic transplants due to immune rejection. HLA-matched stem cell lines may overcome the risk of immune rejection. For parthenogeneic derived cells, HLA heterozygous cell lines are derived from HLA heterozygous donors by activating oocytes using a combination of A23187 and 6-DMAP. Since these cells are HLA-matched with the oocyte donor, their ability to provide tissue-matched derivatives is limited.
MHC compatibility between a donor and recipient increases significantly if the donor cells are HLA homozygous; i.e. contain identical alleles for each antigen-presenting protein. Furthermore, if homozygous donor cells have a haplotype found with high frequency in a population, these cells may have application in transplantation-based stem cell therapies for a large number of individuals.